Pancreatic Cancer (PC) is an extremely deadly disease with a mortality rate of nearly 95%. The current treatment options available for PC patients only extend their lives by a few months. Therefore, there is a critical need to identify potent compounds that could enhance the effectiveness of chemotherapies, especially for K- ras mutant PC cells that are often resistant to treatment. The goal of this research project is to evaluate a novel natural product-based agent in combination with gemcitabine (GEM) for pancreatic cancer therapy, eventually leading to the ultimate development of clinically-useful natural product-based agents for the treatment of cancer. The project objective is to establish piperlongumine (PPLGM) as a chemosensitizer for PC by determining its mechanisms of action and therapeutic efficacy. Our central hypothesis is that PPLGM, isolated from the fruits of long peppers, sensitizes tumor cells, but not normal cells, to chemotherapy-induced cell death through induction of reactive oxygen species (ROS) and enhanced DNA damage. We have formulated this hypothesis based on the existing literature and our own preliminary findings that show PPLGM elevates ROS levels in cancer cells leading to enhanced tumor cell death. To test our central hypothesis, we propose three Specific Aims: 1) To investigate the mechanisms by which PPLGM enhances ROS levels and induces cell death in PC cells; 2) To evaluate the effect of PPLGM on sensitizing PC cells, but not normal cells, to chemotherapy; and 3) To evaluate the therapeutic efficacy of PPLGM alone or in combination with GEM in mouse models of PC. For the first aim, we will treat normal and PC cells that are K-ras mutant and wildtype with PPLGM, and we will determine the molecular mechanisms by which PPLGM causes ROS-induced cell death. For the second aim, we will use the same cells to determine the combined effect of PPLGM and GEM on PC cell death and investigate their synergistic mechanisms for inducing PC cell death. For the third aim, we will evaluate the therapeutic efficacy of PPLGM alone or in combination with GEM using an orthotopic mouse model of K-ras mutant human PC and a transgenic mouse model. The approach uses a novel dietary agent that shows potent anti-cancer effects in the context of chemotherapy for PC. The proposed research is expected to vertically advance and expand understanding of how a ROS-inducing agent can be used as a chemosensitizer for cancer treatment. Ultimately such knowledge has the potential to change the field of chemotherapy and result in more effective treatment for many cancers.